OBJECTIVE

The purpose of this study was to identify the underlying molecular genetic defect in an Indonesian family with three affected individuals who had received a diagnosis of retinitis pigmentosa (RP).

METHODS

Clinical evaluation of the family members included measuring visual acuity and fundoscopy, and assessing visual field and color vision. Genomic DNA of the three affected individuals was analyzed with Illumina 700k single nucleotide polymorphism (SNP) arrays, and homozygous regions were identified using PLINK software. Mutation analysis was performed with sequence analysis of the retinitis pigmentosa 1 (<em>RP</em>1) gene that resided in one of the homozygous regions. The frequency of the identified mutation in the Indonesian population was determined with TaqI restriction fragment length polymorphism analysis.

RESULTS

A novel homozygous nonsense mutation in exon 4 of the RPT (p.R338*), was identified in the proband and her two affected sisters. Unaffected family members either carried two wild-type alleles or were heterozygous carriers of the mutation. The mutation was not present in 184 Indonesian control samples.

CONCLUSIONS

Most of the previously reported RPRPRPRP.

A new PLA2 (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE approximately 14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed Km 6.0 mM, Vmax 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 degrees C, and it had a strict requirement of Ca2+ for its activity. The N-terminal sequence of PLA2 was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA2 from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA2 from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD50 than crotoxin complex from C d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 microg/l ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armcc.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armcce of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armccompared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh performance liquid chromatography tandem mass spectrometry, IP-RP-UHPLC-MS/MS, was developed for quantitative determination of intracellular coenzyme A (CoA), acetyl CoA, succinyl CoA, phenylacetyl CoA, flavin mononucleotide, (FMN), flavin adenine dinucleotide, (FAD), NAD, NADH, NADP, NADPH. Dibutylammonium acetate (DBAA) was used as volatile ion pair reagent in the mobile phase. Addition of DBAA to the sample solutions resulted in an enhanced sensitivity for the phosphorylated coenzymes. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), was added to keep CoA in the reduced form. Isotope dilution mass spectrometry (IDMS) was applied for quantitative measurements for which culture derived global U-(13)C-labeled cell extract was used as internal standard. The analytical method was validated by determining the limit of detection, the limit of quantification, repeatability and intermediate precision. The method was successfully applied for quantification of coenzymes in the cell extracts of Saccharomyces cerevisiae.

The metal removal capacity of cultures of two capsulated, exopolysaccharide-producing cyanobacteria, <em>C</em>yanospira capsulata and Nostoc P<em>C</em><em>C</em>7936, were tested using copper (II) as the model metal. <em>C</em>. capsulata cultures removed the greatest amount of copper, with a maximum per unit of biomass (q(max)) of 115.0+/-5.1 mg copper g(-1) of protein, compared with 85.0+/-3.2 removed with Nostoc P<em>C</em><em>C</em>7936 cultures. Water solutions of pure polysaccharides (<em>RPS</em>s) released into the culture medium by <em>C</em>. capsulata and Nostoc P<em>C</em><em>C</em>7936 achieved q(max) values of 20.2+/-0.8 mg g(-1) copper per polysaccharide dry weight with <em>C</em>. capsulata <em>RPS</em> and 11.0+/-1.5 mg g(-1) with Nostoc P<em>C</em><em>C</em>7936 <em>RPS</em>. <em>C</em>ultures of the two cyanobacteria also removed Zn (II) and Ni (II), in both single-metal systems and in multimetal systems with <em>C</em>u; in the various single-metal systems more copper was removed than Zn or Ni, while in the multimetal systems a smaller amount of each individual metal was removed but the overall amount of all metal ions sorbed or the amount of copper sorbed in the copper-only system was almost the same with <em>C</em>. capsulata, and slightly higher with Nostoc P<em>C</em><em>C</em>7936.

High dietary fructose causes insulin resistance syndrome (IRS), primarily due to simultaneous induction of genes involved in glucose, lipid and mitochondrial oxidative metabolism. The present study evaluates effect of a hepatoprotective agent, silymarin (SYM) on fructose-induced metabolic abnormalities in the rat and also assessed the associated thrombotic complications. Wistar rats were kept on high fructose (HFr) diet throughout the 12-week study duration (9 weeks of HFr feeding and subsequently 3 weeks of HFr plus SYM oral administration [once daily]). SYM treatment significantly reduced the HFr diet-induced increase expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α/β, peroxisome proliferator-activated receptor (PPAR)-α, forkhead box protein O1 (FOXO1), sterol regulatory element binding protein (SREBP)-1c, liver X receptor (LXR)-β, fatty acid synthase (FAS) and PPARγ genes in rat liver. SYM also reduced HFr diet mediated increase in plasma triglycerides (TG), non-esterified fatty acids (NEFA), uric acid, malondialdehyde (MDA), total nitrite and pro-inflammatory cytokines (C-reactive protein [CRP], interleukin-6 [IL-6], interferon-gamma [IFN-γ] and tumor necrosis factor [TNF]) levels. Moreover, SYM ameliorated HFr diet induced reduction in glucose utilization and endothelial dysfunction. Additionally, SYM significantly reduced platelet activation (adhesion and aggregation), prolonged ferric chloride-induced blood vessel occlusion time and protected against exacerbated myocardial ischemia reperfusion (MI-RP) injury. SYM treatment prevented HFr induced mRNA expression of hepatic PGC-1α/β and also its target transcription factors which was accompanied with recovery in insulin sensitivity and reduced propensity towards thrombotic complications and aggravated MI-RP injury.

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.

Slowing down translation in either the cytosol or the mitochondria is a conserved longevity mechanism. Here, we found a non-interventional natural correlation of mitochondrial and cytosolic ribosomal proteins (RPs) in mouse population genetics, suggesting a translational balance. Inhibiting mitochondrial translation in C. elegans through mrps-5 RNAi repressed cytosolic translation. Transcriptomics integrated with proteomics revealed that this inhibition specifically reduced translational efficiency of mRNAs required in growth pathways while increasing stress response mRNAs. The repression of cytosolic translation and extension of lifespan from mrps-5 RNAi were dependent on atf-5/ATF4 and independent from metabolic phenotypes. We found the translational balance to be conserved in mammalian cells upon inhibiting mitochondrial translation pharmacologically with doxycycline. Lastly, extending this in vivo, doxycycline repressed cytosolic translation in the livers of germ-free mice. These data demonstrate that inhibiting mitochondrial translation initiates an atf-5/ATF4-dependent cascade leading to coordinated repression of cytosolic translation, which could be targeted to promote longevity.

A novel monocyte chemotactic factor, a cross-linked homodimer of S19 ribosomal protein (RP S19) was initially isolated from a rheumatoid arthritis synovial lesion. The RP S19 dimer causes the monocyte specific chemotaxis in vitro and the monocyte predominant infiltration in vivo, via its agonistic and antagonistic effects on the CRP S19 and CCC-terminal portion of RP S19. The RP S19 dimer is produced and released by apoptotic cells, and this dimer recruits monocytes from the circulation to the apoptotic lesion. The infiltrated monocytes/macrophages engulf the apoptotic cells, translocate to regional lymph nodes via lymphatics and present the antigenic information of the apoptotic cells to the T cell repertoire. In this manner, the apoptotic cell clearance system connects to the acquired immune system. The innate and acquired immune mechanisms, mediated by the RP S19 dimer, participate in the pathology of inveterate chronic inflammation such as rheumatoid arthritis.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

C-reactive protein (C-RP) determinations were performed by using the latex slide agglutination test on cerebrospinal fluid (CSF) from 235 patients. The patients were categorized into the following groups: bacterial meningitis (n = 74); viral meningitis (n = 10); fever without bacterial meningitis (n = 80); neurological symptoms without infection (n = 25); intracranial hemorrhage (n = 10); increased intracranial pressure that was secondary to pseudotumor cerebri or hydrocephalus (n = 16); and malignancies (n = 20). On the initial lumbar puncture, the C-RP was positive in 97% (72 of 74) of the patients in group 1, as compared with 0% (0 of 10), 6% (5 of 80), 20% (5 of 25), 50% (5 of 10), 6% (1 of 16), and 30% (6 of 20) in groups 2-7, respectively (P less than .0001). The C-RP test was able to detect bacterial meningitis with a sensitivity of 97% (72 of 74), a specificity of 86% (139 of 161), a positive predictive value of 77% (72 of 94), and a negative predictive value of 99% (139 of 141). These data indicate that C-RP determinations performed on CSF are useful and rapid clinical tests for the exclusion of the presence of bacterial meningitis in a patient.

A group of 123 individuals with rheumatoid arthritis (RA group) and 28 individuals with osteoarthrosis (OA group) were compared with 52 individuals without general joint symptoms (C group). The individuals in the RA and OA groups constituted 35% and 14%, respectively, of all individuals with RA and OA at the Rheumatism Hospital in Strängnäs, Sweden, during the period of investigation. All individuals answered a questionnaire concerning subjective symptoms from the stomatognathic system and general joint symptoms and were given a clinical examination comprising the temporomandibular joint (TMJ), masticatory muscles, mandibular mobility, and occlusion. Most signs of disorder in the stomatognathic system were more frequent and more severe in the RA and OA groups than in the C group. The clinical signs were of a similar character in the RA and OA groups, but the individuals in the RA and OA groups had less occlusal support, more occlusal interferences, greater distance between RP and IP, and less vertical overbite than the C group. Anterior open bite was found with higher frequency and severity in the RA and OA groups than in the C group and was correlated to clinical dysfunction score and reduced maximum mouth opening capacity.

OBJECTIVE

To study the relationship between health-related quality of life (HRQOL) and mode of acquisition, treatment discontinuations, drop in haemoglobin levels and treatment outcome in patients with chronic hepatitis C (CHC).

METHODS

Consecutive unselected Swedish patients with CHC completed the SF-36 questionnaire before, during and after treatment with interferon and ribavirin. Results. At baseline, HRQOL was reduced in all SF-36 subscales in our patients (n=147) as compared with the general Swedish population. Former intravenous drug users (IVDUs) scored significantly lower in social function (p=0.03) and mental health (p=0.03) than patients who had acquired their infection from blood transfusions (PTH). A decline of >40 points in HRQOL from baseline to week 12 was noticed in the role limitations-physical (RP) score for the IVDU and PTH groups (p<0.0001 and 0.001, respectively). Patients with a>>or=20% fall in haemoglobin levels at treatment week 12 had a significantly poorer RP (p=0.006) and role limitations-emotional score (p<0.02) than patients with a <10% fall. Early treatment dropouts had significantly lower HRQOL scores at baseline than adherent patients. At follow-up, sustained viral responders had significantly higher scores than non-responders.

CONCLUSIONS

Swedish outpatients with CHC have a marked reduction in their HRQOL as compared to the general population. Therapy reduces HRQOL most substantially in those with a marked reduction in haemoglobin. Early dropouts from therapy have significantly lower HRQOL scores at baseline than adherent patients, and sustained viral responders improve their HRQOL significantly more than non-responders.

Nickel-titanium (NiTi) wires produced by various manufacturers may have different corrosion resistance in acidic oral environments. The purpose of this study was to investigate the variation during in vitro corrosion resistance of commercial NiTi dental orthodontic wires from different manufacturers using the fast electrochemical technique. The linear polarization test was used to evaluate the corrosion resistance, in terms of polarization resistance (Rp), of as-received commercial NiTi wires in acidic artificial saliva at 37 degrees C. One-way analysis of variance was used to analyze Rp with the wire manufacturer as the variable factor. Atomic force microscopy was used to analyze the three-dimensional surface topography and roughness (Ra). Electron spectroscopy for chemical analysis was used to identify the chemical structure of the passive film on the NiTi wires. The results showed that NiTi wires from different manufacturers had a statistically significant difference in Rp (P < .001). Different surface topography was present among the tested NiTi wires, whereas the same surface chemical structure was observed for the tested NiTi wires. The surface roughness of the commercial NiTi wires with similar surface chemical structure does not correspond with the difference in corrosion resistance.

In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of DNA polymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The proteins and enzymatic activities thus far identified to co-purify with the leukemia cell DNA synthesome include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), and DNA topoisomerases I and II. We have demonstrated that the DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all of the cellular activities required for large T-antigen-dependent in vitro SV40 DNA synthesis are present in the isolated human leukemia cell DNA synthesome. Since SV40 is extensively dependent on the host cell's DNA synthetic machinery for its own DNA replication, our results indicate that the isolated leukemia cell DNA synthesome may play a role not only in viral DNA synthesis but also in human leukemia cell DNA replication. We recently proposed a model to represent the DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the DNA synthesome from HL-60 cells also fits this proposed model. The purified DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's DNA synthetic machinery.

Since the description of human T-cell leukemia virus type I (HTLV-I) and its simian counterpart, simian T-cell leukemia virus type I (STLV-I), the possible existence of other related simian retroviruses has been raised. Here, we report a new retrovirus, STLV-II, which we have identified in spider monkeys (Ateles fusciceps), a New World primate species. Initially, a recombinant HTLV-II envelope protein (RP-IIB) was used to identify anti-STLV-II antibodies in New World monkeys by Western blot (immunoblot) assays. Subsequently, the virus was characterized by Southern blot hybridization, which showed that STLV-II and HTLV-II have a high degree of nucleotide sequence homology but have different restriction enzyme patterns. Nucleotide sequence analysis of the pX-II region of STLV-II provirus revealed 3% variation with the corresponding region of HTLV-II. Electron micrographic studies revealed HTLV-like, type C retrovirus particles outside the cell membranes of STLV-II-infected cells. This study describes the first link between HTLV-II and a simian reservoir in the New World. Further molecular studies of STLV-II infection in different species of New World monkeys, especially from the wild, may provide valuable information about the origin and intragroup relationships of South American monkeys. Spider monkeys infected with STLV-II may serve as an important animal model for HTLV-II infection in humans.

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPCCF cells.

The stability of ascorbic acid, ascorbyl palmitate and magnesium ascorbyl phosphate (VC-PMG) in both standard solutions and topical formulations was investigated by direct RP-HPLC analysis after sample dilution with a suitable aqueous-organic solvent mixture. The results showed that, whereas the two vitamin C derivatives were more stable than ascorbic acid, the ascorbyl esters showed significant differences. Esterification with palmitic acid in 6 position did not prevent hydrolysis of the molecule, either in solution or in emulsion; only the special preparation of products with high viscoelastic properties was able to reduce the typical behaviour of this compound. Conversely, the introduction of the phosphoric group in 2 position protected the molecule from break-up of the enediol system, confirming VC-PMG as a very stable derivative of vitamin C that may be easily used in various types of cosmetic products.

To estimate the heritability (h2) of postrigor calpastatin activity (CA), 555 steers were reared and processed conventionally. Breed-types included purebreds (Angus [A], Braunvieh [B], Charolais [C], Gelbvieh [G], Hereford [H], Limousin [L], Pinzgauer [P], Red Poll [RP], and Simmental [S]), composite populations (MARC I [1/4 C, 1/4 B, 1/4 L, 1/8 H, 1/8 A], MARC II [1/4 S, 1/4 G, 1/4 H, 1/4 A], and MARC III [1/4 RP, 1/4 H, 1/4 P, 1/4 A]), and F1 crosses (H, A, C, G, P, Shorthorn, Galloway, Longhorn, Nellore, Piedmontese, or Salers x H or A). Steers were serially slaughtered on an age-constant (across breed groups) basis. Heritability estimates for CA, i.m. fat content (IMF), Warner-Bratzler shear (WBS) force, retail product yield (RPY), and ADG were .65 +/- .19, .93 +/- .02, .53 +/- .15, .45 +/- .18, and .32 +/- .26, respectively. The genetic correlations (rg) of CA with WBS, RPY, and ADG were .50 +/- .22, .44 +/- .25, and -.52 +/- .37, respectively. The rg of IMF with WBS, RPY, and ADG were -.57 +/- .16, -.63 +/- .15, and -.04 +/- .11, respectively. These h2 and rg estimates indicate that it should be possible to select for improvements in CA, IMF, and WBS. However, selection against CA may be a more suitable approach for improving meat tenderness than selection for increased IMF because the level of genetic antagonism between CA and RPY was not as great as that between IMF and RPY.

The present study was undertaken to investigate whether RP-1 treatment protected mitochondrial system against radiation damage and also to unravel the mechanism associated with this process. Radioprotection of mitochondrial system by Podophyllum hexandrum (RP-1) was investigated to understand its mechanism of action. Levels of superoxide anion (O2-), reduced or oxidized glutathione (GSH or GSSG), thiobarbituric acid reactive substance (TBARS), protein carbonyl (PC), ATP, NADH-ubiquinone oxidoreductase (complex-I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (complex II/III) and mitochondrial membrane potential (MMP) were studied in mitochondria isolated from liver of mice belonging to various treatment groups. Whole body y-irradiation (10 Gy) significantly (p < 0.01) increased the formation of O2-, PC, and TBARS, upto 24 h as compared to untreated control. RP-1 treatment (200 mg/kg b.w.) to mice 2 h before irradiation reduced the radiation-induced O2- generation within 4 h and formation of TBARS and PC upto 24 h significantly (p < 0.01). Singularly irradiation or RP-1 treatment significantly (p < 0.01) increased the levels of glutathione within an hour, as compared to untreated control. Pre-irradiation administration of RP-1 enhanced levels of GSH induced increase in complex I (upto 16 h), complex I/III (4 h) complex II/III activity (upto 24 h; p < 0.01) and inhibited the radiation-induced decrease in MMP significantly (24 h; p < 0.01). The present study indicates that RP-1 itself modulates several mitichondrial perameters due to its influence on the biochemical milieu within and outside the cells. However, RP-1 treatment before irradiation modulates radiation induced perturbations such as the increase in electron transport chain enzyme activity, formation of O2-, TBARS and PC to offer radioprotection.

BACKGROUND

Uroguanylin is an endogenous peptide agonist that binds to the guanylate cyclase C receptor (GC-C). GC-C is overexpressed in human colorectal cancer (CRC), and exposure of GC-C-expressing cells to GC-C agonists results in cell cycle arrest and/or apoptosis, highlighting the therapeutic potential of such compounds. This study describes the first use of radiolabeled uroguanylin analogs for in vivo detection of CRC.

METHODS

The peptides uroguanylin and E(3)-uroguanylin were N-terminally labeled with the DOTA chelating group via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with In-111 and used for in vivo biodistribution and SPECT imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts.

RESULTS

Alteration of the position 3 aspartate residue to glutamate resulted in increased affinity for GC-C, with IC(50) values of 5.0+/-0.3 and 9.6+/-2.9 nM for E(3)-uroguanylin and DOTA-E(3)-uroguanylin, respectively. In vivo, (111)In-DOTA-E(3)-uroguanylin demonstrated tumor uptake of 1.17+/-0.23 and 0.61+/-0.07% ID/g at 1 and 4 h post injection, respectively. The specificity of tumor localization was demonstrated by coinjection of 3 mg/kg unlabeled E(3)-uroguanylin, which reduced tumor uptake by 69%. Uptake in kidney, however, was dramatically higher for the uroguanylin peptides than for previously characterized radiolabeled E. coli heat-stable enterotoxin (STh) analogs targeting GC-C, and was also inhibited by coinjection of unlabeled peptide in a fashion not previously observed.

CONCLUSIONS

Use of uroguanylin-targeting vectors for in vivo imaging of colorectal cancers expressing GC-C resulted in tumor uptake that paralleled that of higher affinity heat-stable enterotoxin peptides, but also resulted in increased kidney uptake in vivo.

Phosphorylation of 2',3'-O-methoxymethylidene adenosine with bis(p-nitrophenyl) O,O-phosphorochloridothioate, followed by alkaline and acidic deprotection, afforded a mixture of the Rp and Sp diastereomers of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester in good yield. Only one of these diastereomers is a substrate for snake venom phosphodiesterase. The other diastereomer, remaining after complete enzymatic digestion of the substrate, was crystallized as the triethylammonium salt in the triclinic space group P1. The structure was solved from three-dimensional X-ray data and refined to an R value of 5.2%. The asymmetric cell unit contains two independent molecules with almost identical conformations. The ribose is puckered C(2')-endo, the heterocycle is in anti position and the C(5')-O(5') bond gauche, gauche. The triethylammonium cation is coordinated to the free oxygen of the phosphorothioate group and the P-S bond has double bond character. The absolute configuration of the phosphorous is Sp. This is in agreement with an earlier stereochemical study of the hydrolysis of the diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate by snake venom phosphodiesterase.

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on the L-type Ca2+ channel current (L-channel current) was studied in smooth muscle cells prepared from the rat tail artery. PACAP caused an increase in the amplitude of the L-channel current. The maximal increase (56%) occurred at a PACAP concentration of 1 x 10(-8) M; higher concentrations resulted in a smaller increase. Investigation into the intracellular mechanisms of PACAP action revealed that the increase in L-channel currents was blocked by calphostin C and bisindolylmaleimide IV [protein kinase C (PKC) inhibitors] and mimicked by 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PACAP was also found to cause translocation of PKC, suggesting that the increase in the current by PACAP was due to PKC. In contrast, activation of cAMP-dependent protein kinase (PKA) by 8-bromo-cAMP caused an inhibition of the L-channel current. A high concentration of PACAP (1 x 10(-6) M) had no effect on the L-channel current. The null effect of PACAP on the L-channel current could be converted to an increase by Rp-cAMPs, a cAMP antagonist, and a decrease by calphostin C. PACAP also increased cAMP accumulation. These observations indicate the effect of PACAP on the L-channel current represents the integration of two signaling mechanisms that involve the activation of PKA and PKC.